Research Summary Recombination between chromosomes is required to generate genetic variation, maintain genome integrity through the repair of double strand DNA breaks (DSBs), and ensure proper chromosome segregation during meiosis, the specialized cell division program by which diploid organisms generate haploid gametes such as sperm and eggs. Perturbations in recombination can compromise these basic cellular functions, ultimately leading to cancer, infertility, or birth defects. Meiotic recombination is initiated by DSBs, which are repaired using meiosis-specific mechanisms that favor utilization of the homologous chromosome (instead of the sister chromatid) as the recombination partner and that promote a crossover outcome of the DSB repair process, which is required for promoting proper chromosome segregation during meiosis. Although repair of DSBs with the appropriate template (homologous chromosome) is necessary for proper chromosome segregation and genome integrity, our knowledge about how germ cells achieve this template preference in the presence of nearly identical sequences (sister chromatids) is limited. The goal of our research program is to understand how chromosomes are able to access distinct recombination pathways and partners depending on the chromosomal and cellular context to ensure faithful genome inheritance. Using Caenorhabditis elegans as a model system, we have developed an assay to monitor repair of an induced DSB with the sister chromatid during meiotic prophase progression in vivo. We now have evidence for the presence of a switch in repair partner preferences from the homologous chromosome to the sister chromatid during late meiotic prophase. In this proposal we will begin to lay the foundation for understanding how specific DSB repair pathways and partners are engaged and regulated during meiosis. To determine how these different repair partner choices are regulated, we will identify the molecular signatures, chromosomal features, and proteins associated with these different repair outcomes. Further, we will use live imaging, a new live genomic locus tracking system, and genetics to determine the how early DSB repair dynamics influence DSB repair outcomes. Further, we will use superresolution and high-resolution microscopy methods to assess the effects of DSB repair on meiotic chromosome structures and DNA organization. Overall, these studies will reveal how recombination pathway and partner choices ensure that chromosomes form the connections necessary for chromosome segregation and repair DSBs for maintaining genomic integrity during sperm and egg development.